The bacteriophage T4 regA protein is a translational repressor of several T4 mRNAs. Genetic methods are described in this proposal that will identify amino acids of the regA protein that contact the mRNA binding sites. In the principle project, plasmids that allow controlled regA expression will be mutagenized in vitro and in vivo and will then be screened for altered regA function in E. coli strains containing regA-sensitive genes or sensitive gene fusions. Kinetic descriptions will be obtained for regA proteins with diminished, tight-binding or new site-specifity phenotypes that will then be correlated to the single amino acid substitution (determined by nucleic acid sequencing) in each repressor mutant. Amino acid residues and short regions of regA have been selected for targeted mutagenesis. From the initial results using random mutagenesis and the described new selection techniques, regA residues of particular interest will be identified for further directed mutagenesis studies. Two aspects of regA function are addressed as a smaller effort of this proposal. A translational activation function has been tentatively ascribed to the regA protein; the activated proteins will be identified by 2D gel electrophoresis and sequenced. The regA mRNA binding site will also be characterized by random oligonucleotide synthesis in a collaborative effort with an established laboratory working on translational regulation. These efforts will complement our principle interests described above. The long-term goal of the laboratory is to define the RNA-protein interactions of the regA translational repressor with the expectation that features of regA will be relevant to understanding RNA-protein interactions of other prokaryotic and eukaryotic systems.